The ligation of polymerized nucleic acids is a necessary step in the fundamental techniques of molecular biology. Ligation of deoxyribonucleic acid (DNA) polymers is ubiquitous in cloning, sequencing and analyzing genetic material. There are several general texts which describe ligation generally, including Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook").
Techniques utilizing ligation are becoming increasingly important as general tools of basic research and in clinical settings. The ligation chain reaction (LCR; sometimes denoted the "ligation amplification reaction" or "LAR") and related techniques are of increasing utility, particularly as diagnostic tools. LCR (described below) provides a mechanism for linear or exponential amplification of a target nucleic acid via ligation of adjacent oligonucleotides. This amplification may be performed to distinguish target nucleic acids which differ by a single nucleotide, providing a powerful tool for the diagnosis of genetic disease, the analysis of genetic variation, and the detection of specific DNA sequences. A problem of ligation generally, and of LCR in particular is a lack of specificity in the ligation reaction. Undesired ligation products increase the percentage of undesired constructs to desired constructs or "noise" in the cloning process, and increase the noise to signal ratio in LCR, limiting the analytic ability of the technique.
The present invention overcomes the limitations of the prior art by increasing substantially the specificity of ligation. The increase in specificity is achieved by substitution of deoxyribose NAD.sup.+ for NAD.sup.+ as a cofactor in NAD.sup.+ -dependent ligation reactions. Examples of ligase enzymes used in ligation reactions which require NAD.sup.+ as a cofactor include E. coli DNA ligase and Taq DNA ligase, each of which displays an increase in the specificity of ligation upon the addition of .beta.-2'-deoxyribose NAD.sup.+ or .beta.-3'-deoxyribose NAD.sup.+.